Recurrence and metastatic dissemination of breast cancers account for a significant morbidity and mortality in women, and effective means of treating this subset of patients remains elusive. In his post-doctoral studies, John Ojeifo, M.D., Ph.D., developed a novel t h e rapeutic drug delivery system that utilizes genetically-engineered endothelial cells to treat a variety of diseases. In this Mentored Career Development Award application, the candidate outlines a plan that will enable him to mature into a fully independent investigator studying the therapeutic efficacy and safety of intravenously (IV)-administered, genetically-modified endothelial cells (GMECs) transduced to express a cytokine and a suicide transgenes in metastatic breast cancer-bearing mice. During years 1 and 2 of the award, the candidate will benefit from the co-mentorship of Dr. Marc Lippman, a renowned expert in breast cancer biology and treatment, and Dr. Esther Chang, an expert in gene therapy. By year 3, Dr. Ojeifo will be a fully independent investigator. The broad goal of this application is to establish an independent laboratory studying: (I) the molecular and cellular mechanisms contributing to immune dysfunction in human cancers, and (ii) devising novel strategies for effective and safe genetic therapy and prevention of human cancers. To this end, the applicant first plans to test the hypothesis that a mixture of endothelial cells stably expressing herpes simplex virus thymidine kinase (HSV-TK) and h u man interleukin-2 (hIL-2) transgenes can target sites of pulmonary metastasis of breast tumors, induce an anti-tumor immune response at the local site, and abrogate the tumors in mice. The Specific Aims of this proposal are: Aim 1: To construct a bicistronic retroviral vector containing herpes simplex virus thymidine kinase (HSV-TK) and E. coli lacZ genes with intervening IRES fragment, and generate endothelial cells expressing the HSV-TK and lacZ transgenes. Aim 2: To determine (a) How well murine endothelial cells expressing HSV-TK transgene can target metastatic sites of breast tumors, and (b) the effects of HSV-TK gene expression upon tumor metastasis and animal survival. Aim 3: To determine whether the administration of a mixture of hIL-2 genetically-modified endothelial cells (GMECs) and HSV-TK GMECs followed by ganciclovir treatment can induce an effective, specific, and long-term anti-tumor immune response, and abrogate lung metastasis of breast tumors in mice.